Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth
To explore the regulation and function of D-type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth-inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G1; association of D-type cyclins with cyclin-dependent kinase (cdk) 4 and cdk6; G1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D-type cyclins; and association of p16INK4a, p15INK4b, and p27Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G1 arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G1), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4-fold in treated versus untreated cells. At 5 h, cyclin D-associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4-associated p27Kip1 and, at 9 h, a fourfold increase in cdk6-associated p27Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cells but not their Rb-negative parental Du/145 cells. The data suggest that HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4- and cdk6-associated p27Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state.